(A) Schematic diagram of outer retina and Bruch’s membrane. Blue, pink, yellow, and green bands indicate layers formed by highly compartmentalized and vertically aligned photoreceptors and RPE cells in panels B and C. Layers: OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; RPE, retinal pigment epithelium; BrM, Bruch’s membrane; R, Rod; C, cone photoreceptors. (B–F) Overlaid MALDI-MSI images and H&E stained cross-section of the peripheral retina display signals from multiple lipid classes that localize to specific subcellular compartments of the photoreceptor cells and RPE. (B) Overlay showing four separate signals defined in panels C–F, localized to (C) ONL, (D) photoreceptor inner and outer segments, (E) mitochondria-rich photoreceptor inner segments, and (F) RPE apical processes. ©2021 Anderson et al. Reprinted from an article originally published in J. Am. Soc. Mass Spectrom. https://doi.org/10.1021/jasms.0c00119.